Adhesive complex coacervates and methods of making and using thereof

ABSTRACT

Described herein is the synthesis of adhesive complex coacervates. The adhesive complex coacervates are composed of a mixture of one or more polycations, one or more poly anions, and one of more multivalent cations. The polycations and polyanions in the adhesive complex coacervate are crosslinked with one another by covalent bonds upon curing. The adhesive complex coacervates have several desirable features when compared to conventional bioadhesives, which are effective in water-based applications. The adhesive complex coacervates described herein exhibit good interfacial tension in water when applied to a substrate (i.e., they spread over the interface rather than being beaded up). Additionally, the ability of the complex coacervate to crosslink intermolecularly increases the cohesive strength of the adhesive complex coacervate. The adhesive complex coacervates have numerous biological applications as bioadhesives and drug delivery devices. In particular, the adhesive complex coacervates described herein are particularly useful in underwater applications and situations where water is present such as, for example, physiological conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority upon U.S. provisional application Ser.No. 61/023,173, filed Jan. 24, 2008. This application is herebyincorporated by reference in its entirety for all of its teachings.

BACKGROUND

Bone fractures are a serious health concern in society today. Inaddition to the fracture itself, a number of additional health risks areassociated with the fracture. For example, intra-articular fractures arebony injuries that extend into a joint surface and fragment thecartilage surface. Fractures of the cartilage surface often lead todebilitating posttraumatic arthritis. The main determining factors inthe development of posttraumatic arthritis are thought to be the amountof energy imparted at the time of injury, the patient's geneticpredisposition (or lack thereof) to posttraumatic arthritis, and theaccuracy and maintenance of reduction. Of the three prognostic factors,the only factor controllable by orthopedic caregivers is achievement andmaintenance of reduction. Comminuted injuries of the articular surface(the cartilage) and the metaphysis (the portion of the bone immediatelybelow the cartilage) are particularly challenging to maintain in reduced(aligned) position. This relates to the quality and type of bone in thisarea. It also relates to the limitations of fixation with titanium orstainless steel implants.

Currently, stainless steel and titanium implants are the primary methodsof fixation, but their size and the drilling necessary to place themfrequently interfere with the exact manipulation and reduction ofsmaller pieces of bone and cartilage. A variety of bone adhesives havebeen tested as alternatives to mechanical fixation. These fall into fourcategories: polymethylmethacrylates (PMMA), fibrin-based glues, calciumphosphate (CP) cements, and CP resin composites. PMMA cements, which areused in the fixation of protheses, have well-known drawbacks, one of themost serious being that the heat generated from the exothermic settingreaction can kill adjacent bone tissue. Also, the poor bonding to boneleads to aseptic loosening, the major cause of PMMA cemented prothesisfailure.

Fibrin glues, based on the blood clotting protein fibrinogen, have beentested for fixing bone grafts and repairing cartilage since the 1970sand yet have not been widely deployed. One of the drawbacks of fibringlues is that they are manufactured from pooled human donor blood. Assuch, they carry risk of transmitting infections and could potentiallybe of limited supply.

CP cements are powders of one or more forms of CP, e.g., tetracalciumphosphate, dicalcium phosphate anhydride, and β-tricalcium phosphate.When the powder is mixed with water it forms a paste that sets up andhardens through the entanglement of one or more forms of CP crystals,including hydroxyapatite. Advantages of CP cements include isothermalset, proven biocompatibility, osteoconductivity, and they serve as areservoir for Ca and PO₄ for hydroxyapatite formation during healing.The primary disadvantages are that CP cements are brittle, have lowmechanical strength and are therefore not ideal for stable reduction ofsmall articular segments. CP cements are used mostly as bone voidfillers. The poor mechanical properties of CP cements have led tocomposite cements of CP particles and polymers. By varying the volumefractions of the particulate phase and the polymer phase, the modulusand strength of the glue can be adjusted toward those of natural bone,an avenue that is also open to us.

Given the overall health impact associated with bone fractures and theimperfect state of current fixation methods, new fixation methods areneeded.

SUMMARY

Described herein is the synthesis of adhesive complex coacervates. Theadhesive complex coacervates are composed of a mixture of one or morepolycations, one or more polyanions, and one of more multivalentcations. The polycations and polyanions are crosslinked with one anotherby covalent bonds upon curing. The adhesive complex coacervates haveseveral desirable features when compared to conventional adhesives,which are effective in water-based applications. The adhesive complexcoacervates described herein exhibit low interfacial tension in waterwhen applied to a substrate (i.e., they spread over the interface ratherthan being beaded up). Additionally, the ability of the complexcoacervate to crosslink intermolecularly increases the cohesive strengthof the adhesive complex coacervate. The adhesive complex coacervateshave numerous biological applications as bioadhesives and drug deliverydevices. In particular, the adhesive complex coacervates describedherein are particularly useful in underwater applications and situationswhere water is present such as, for example, physiological conditions.

The advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the aspects describedbelow. The advantages described below will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

FIG. 1 shows a model of pH dependent coacervate structure and adhesivemechanisms. (A) The polyphosphate (black) with low charge density pairedwith the polyamine (red) form nm-scale complexes. The complexes have anet positive charge. (B) Extended high charge density polyphosphatesform a network connected by more compact lower charge density polyaminesand when present divalent cations (green symbols). The net charge on thecopolymers is negative. (C) Oxidation of 3,4-dihydroxyphenol (D) by O₂or an added oxidant initiates crosslinking between the quinone (Q) andprimary amine sidechains. The coacervate can adhere to thehydroxyapatite surface through electrostatic interactions,3,4-dihydroxyphenol sidechains, and quinone-mediated covalent couplingto matrix proteins.

FIGS. 2-7 shows several protein sequences produced by P. californicathat can be used as polycations and polyanions in the present inventionas well as synthetic polycations and polyanions useful in the presentinvention.

FIG. 8 shows different mechanisms of DOPA crosslinking.

FIG. 9 shows dual syringe systems for applying small “spot welds” ofcomplex coacervates described herein to repair fractures (A), small boneinjuries (B), or bonding synthetic scaffolds to bony tissue (C).

FIG. 10 shows the structure and UV/VIS characterization of mimeticcopolymers. (A) The Pc3 analog, 1, contained 88.4 mol % phosphate, 9.7mol % dopamide, and 0.1 mol % FITC sidechains. The Pc1 analog, 2,contained 8.1 mol % amine sidechains. The balance was acrylamidesubunits in both cases. (B) A single peak at 280 nm characteristic ofthe catechol form of 3,4-dihydroxyphenol was present in the spectrumof 1. Following oxidation with NaIO₄ a peak at 395 nm corresponding tothe quinone form appeared confirming the expected redox behavior of the3,4-dihydroxyphenol containing polymer.

FIG. 11 shows the pH dependent complex coacervation of mixedpolyelectrolytes. (A) At low pH, a 50 mg/ml mixture of 1 and 2 havingequal quantities of amine and phosphate sidechains formed stablecolloidal PECs. As the pH increased the polymers condensed into a denseliquid complex coacervate phase. At pH 10 the copolymers went intosolution and oxidatively crosslinked into a clear hydrogel. (B) The netcharge of the copolymer sidechains as a function of pH calculated fromthe copolymer sidechain densities. (C) The diameter of the PECs(circles) increased nearly three-fold over the pH range 2-4. Above pH 4the complexes flocculate and their size could not be measured. The zetapotential (squares) was zero near pH 3.6 in agreement with thecalculated net charge.

FIG. 12 shows the liquid character of an adhesive complex coacervate.The solution of 1 and 2 contained equal quantities of amine andphosphate sidechains, pH 7.4.

FIG. 13 shows the phase diagram of polyelectrolytes and divalentcations. The amine to phosphate sidechain and phosphate sidechain todivalent cation ratios were varied at a fixed pH 8.2. The state of thesolutions represented in a gray scale. The mass (mg) of the coacervatephase is indicated in the dark grey squares. The compositions indicatedwith an asterisk were used to test bond strength.

FIG. 14 shows the bond strength, shear modulus, and dimensionalstability of coacervate bonded bones. (A) Bond strength at failureincreased ˜50% and the stiffness doubled as the divalent cation ratiowent from 0 to 0.4 relative to phosphate sidechains. Specimens wetbonded with a commercial cyanoacrylate adhesive were used as areference. (n=6 for all conditions) (B) Bonds of adhered bone specimensfully submerged in PBS for four months (pH 7.2) did not swellappreciably.

FIG. 15 shows UV-vis spectra of dopamine copolymers before and afteroxidation (pH 7.2). A catechol peak present before oxidation wasconverted into the quinone form. Top left: p(DMA[8]-Aam[92]). Bottomleft: p(AEMA[30]-DMA[8]). Right: Hydrogel formation by oxidativecrosslinking of dopamine copolymers. (A) p(DMA[8]-Aam[92]). (B)p(EGMP[92]-DMA[8]). (C) p(DMA[8]-Aam[92]) mixed withp(AEMA[30]-Aam[70]). (D) p(EGMP[92]-DMA[8]) mixed withp(AEMA[30]-Aam[70]). Bracketed numbers indicate mol % of sidechains.Arrows indicate direction spectra are changing over time.

FIG. 16 shows pH dependence of dopamine oxidation inpoly(EGMP[92]-DMA[8]). Arrows indicate direction spectra change withtime. Top: pH 5.0, time course inset. Bottom: pH 6.0.

FIG. 17 shows direct contact of (A) human foreskin fibroblasts, (B)human tracheal fibroblasts, and (C) rat primary astrocytes with adhesive(red auto-fluorescent chunks, white asterisks). Cell morphology,fibronectin secretion, and motility are indistinguishable from cellsgrowing in the absence of glue. Green=intermediate filament proteins.Red=secreted fibronection. Blue=DAPI stained nuclei.

FIG. 18 shows a multi-fragment rat calvarial defect model. (A)Generation of defect. (B) Fragmentation of bone cap. (C) Replacement offragments in defect. (D) Application of bone glue. (E-F) Curing(darkening) of glue. Fragments are firmly fixed in E and F.

FIG. 19 shows the effect of pH and normalized net charge with respect toforming adhesive complex coacervates.

DETAILED DESCRIPTION

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that theaspects described below are not limited to specific compounds, syntheticmethods, or uses as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular aspects only and is not intended to be limiting.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a pharmaceutical carrier” includes mixtures of two or moresuch carriers, and the like.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not. For example, the phrase “optionally substituted lower alkyl”means that the lower alkyl group can or can not be substituted and thatthe description includes both unsubstituted lower alkyl and lower alkylwhere there is substitution.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint.

References in the specification and concluding claims to parts byweight, of a particular element or component in a composition orarticle, denotes the weight relationship between the element orcomponent and any other elements or components in the composition orarticle for which a part by weight is expressed. Thus, in a compoundcontaining 2 parts by weight of component X and 5 parts by weightcomponent Y, X and Y are present at a weight ratio of 2:5, and arepresent in such ratio regardless of whether additional components arecontained in the compound.

A weight percent of a component, unless specifically stated to thecontrary, is based on the total weight of the formulation or compositionin which the component is included.

Variables such as R¹, R², R³, R⁴, R⁵, X, m, and n used throughout theapplication are the same variables as previously defined unless statedto the contrary.

The term “alkyl group” as used herein is a branched or unbranchedsaturated hydrocarbon group of 1 to 25 carbon atoms, such as methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl,heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and thelike. Examples of longer chain alkyl groups include, but are not limitedto, an oleate group or a palmitate group. A “lower alkyl” group is analkyl group containing from one to six carbon atoms.

Any of the compounds described herein can be thepharmaceutically-acceptable salt. In one aspect,pharmaceutically-acceptable salts are prepared by treating the free acidwith an appropriate amount of a pharmaceutically-acceptable base.Representative pharmaceutically-acceptable bases are ammonium hydroxide,sodium hydroxide, potassium hydroxide, lithium hydroxide, calciumhydroxide, magnesium hydroxide, ferrous hydroxide, zinc hydroxide,copper hydroxide, aluminum hydroxide, ferric hydroxide, isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine,ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine,arginine, histidine, and the like. In one aspect, the reaction isconducted in water, alone or in combination with an inert,water-miscible organic solvent, at a temperature of from about 0° C. toabout 100° C. such as at room temperature. In certain aspects whereapplicable, the molar ratio of the compounds described herein to baseused are chosen to provide the ratio desired for any particular salts.For preparing, for example, the ammonium salts of the free acid startingmaterial, the starting material can be treated with approximately oneequivalent of pharmaceutically-acceptable base to yield a neutral salt.

In another aspect, if the compound possesses a basic group, it can beprotonated with an acid such as, for example, HCl, HBr, or H₂SO₄, toproduce the cationic salt. In one aspect, the reaction of the compoundwith the acid or base is conducted in water, alone or in combinationwith an inert, water-miscible organic solvent, at a temperature of fromabout 0° C. to about 100° C. such as at room temperature. In certainaspects where applicable, the molar ratio of the compounds describedherein to base used are chosen to provide the ratio desired for anyparticular salts. For preparing, for example, the ammonium salts of thefree acid starting material, the starting material can be treated withapproximately one equivalent of pharmaceutically-acceptable base toyield a neutral salt.

Described herein are adhesive complex coacervates and their applicationsthereof. In general, the complexes are a mixture of cations and anionsin balanced proportions to produce stable aqueous complexes at a desiredpH. The adhesive complex coacervate comprises at least one polycation,at least one polyanion, and at least one multivalent cation, wherein atleast one polycation or polyanion is a synthetic compound, and thepolycation and/or polyanion are crosslinked with one another upon curingthe complex coacervate. Each component of the coacervate and methods formaking the same are described below.

The adhesive complex coacervate is an associative liquid with a dynamicstructure in which the individual polymer components diffuse throughoutthe entire phase. Complex coacervates behave rheologically like viscousparticle dispersions rather than a viscoelastic polymer solution. Asdescribed above, the adhesive complex coacervates exhibit lowinterfacial tension in water when applied to substrates either underwater or that are wet. In other words, the complex coacervate spreadsevenly over the interface rather than being beading up. Additionally,upon intermolecular crosslinking, the adhesive complex coacervate formsa strong, insoluble, cohesive material.

Conversely, polyelectrolyte complexes (PECs), which can be a precursorto the adhesive complex coacervates described herein, are smallcolloidal particles. For example, referring to FIG. 11A, a solution ofPECs at pH 3.1 and 4.2 exists as a milky solution of colloidal particleshaving a diameter of about 300 nm Upon raising the pH to 7.2 and 8.1,the PEC condenses into a liquid phase of concentrated polymers (thecoacervate phase) and a dilute equilibrium phase. In this aspect, thePEC can be converted to an adhesive complex coacervate described herein.

An exemplary model of the differences in phase behavior between thepolyelectrolyte complex and the adhesive complex coacervate is presentedin FIG. 1. At low pH the oppositely charged polyelectrolytes associateelectrostatically into nano-complexes with a net positive surface chargethat stabilizes the suspension to produce PEC 1. With increasing pH thenet charge of the complexes changes from positive to negative butremains near net neutrality. The PEC can form a loose precipitate phase,which can be converted to a complex coacervate 2 by raising the pHfurther (FIG. 1). Thus, in certain aspects, the conversion of the PEC tocomplex coacervate can be “triggered” by adjusting the pH and/or theconcentration of the multivalent cation. For example, the PEC can beproduced at a pH of less than or equal to 4, and the pH of the PEC canbe raised to greater than or equal to 7.0, from 7.0 to 9.0, or from 8.0to 9.0 to convert the PEC to a complex coacervate. Subsequentcrosslinking between the polycation and polyanions (e.g., oxidation andcovalent crosslinking as shown in FIG. 1C) results in the formation ofthe adhesive complex coacervate described herein.

The polycations and polyanions contain groups that permit crosslinkingbetween the two polymers upon curing to produce new covalent bonds andthe adhesive complex coacervate described herein. The mechanism ofcrosslinking can vary depending upon the selection of the crosslinkinggroups. In one aspect, the crosslinking groups can be electrophiles andnucleophiles. For example, the polyanion can have one or moreelectrophilic groups, and the polycations can have one or morenucleophilic groups capable of reacting with the electrophilic groups toproduce new covalent bonds. Examples of electrophilic groups include,but are not limited to, anhydride groups, esters, ketones, lactams(e.g., maleimides and succinimides), lactones, epoxide groups,isocyanate groups, and aldehydes. Examples of nucleophilic groups arepresented below.

In one aspect, the crosslinkable group includes a hydroxyl-substitutedaromatic group capable of undergoing oxidation in the presence of anoxidant. In one aspect, the hydroxyl-substituted aromatic group is adihydroxyphenol or halogenated dihydroxyphenol group such as, forexample, DOPA and catechol (3,4 dihydroxyphenol). For example, in thecase of DOPA, it can be oxidized to dopaquinone. Dopaquinone is anelectrophilic group that is capable of either reating with a neighboringDOPA group or another nucleophilic group. In the presence of an oxidantsuch as oxygen or other additives including, but not limited to,peroxides, periodates, or transition metal oxidants (e.g., NaIO₄ or aFe⁺³ compound), the hydroxyl-substituted aromatic group can be oxidized.In another aspect, crosslinking can occur between the polycation andpolyanion via light activated crosslinking through azido groups. Onceagain, new covalent bonds are formed during this type of crosslinking.

The stability of the oxidized crosslinker can vary. For example, thephosphono containing polyanions described herein that contain oxidizablecrosslinkers are stable in solution and do not crosslink withthemselves. This permits nucleophilic groups present on the polycationto react with the oxidized crosslinker. This is a desirable feature ofthe invention, which permits the formation of intermolecular bonds and,ultimately, the formation of a strong adhesive. Examples of nucleophilicgroups that are useful include, but are not limited to, hydroxyl, thiol,and nitrogen containing groups such as substituted or unsubstitutedamino groups and imidazole groups. For example, residues of lysine,histidine, and/or cysteine can be incorporated into the polycation andintroduce nucleophilic groups. An example of this is shown in FIG. 8.DOPA residue 1 can be oxidized to form a dopaquinone residue 2.Dopaquinone is a reactive intermediate and can crosslink (i.e., react)with a DOPA residue on another polymer or the same polymer to produce adi-DOPA group. Alternatively, the dopaquinone residue can react withnucleophiles such as, for example, amino, hydroxyl, or thiol groups viaa Michael-type addition to form a new covalent bond. Referring to FIG.8, a lysyl group, cysteinyl group, and histidyl group react with thedopaquinone residue to produce new covalent bonds. Although DOPA is asuitable crosslinking group, other groups such as, for example, tyrosinecan be used herein. The importance of crosslinking with respect to theuse of the adhesive complex coacervates described herein will bediscussed below.

In other aspects, the crosslinkers present on the polycation and/orpolyanion can form coordination complexes with transition metal ions.For example, a transition metal ion can be added to a mixture ofpolycation and polyanion, where both polymers contain crosslinkerscapable of coordinating with the transition metal ion. The rate ofcoordination and dissociation can be controlled by the selection of thecrosslinker, the transition metal ion, and the pH. Thus, in addition tocovalent crosslinking as described above, crosslinking can occur throughelectrostatic, ionic, or other non-covalent bonding. Transition metalions such as, for example, iron, copper, vanadium, zinc, and nickel canbe used herein.

The polycation and polyanion are generally composed of a polymerbackbone with a plurality of chargeable groups at a particular pH. Thegroups can be pendant to the polymer backbone and/or incorporated withinthe polymer backbone. The polycation is any biocompatible polymerpossessing cationic groups or groups that can be readily converted tocationic groups by adjusting the pH. In one aspect, the polycation is apolyamino compound. The amino group can be branched or part of thepolymer backbone. The amino group can be a primary, secondary, ortertiary amino group that can be protonated to produce a cationicammonium group at a selected pH. For example, the amino group can bederived from a residue of lysine, histidine, or imidazole attached tothe polycation. Any anionic counterions can be used in association withthe cationic polymers. The counterions should be physically andchemically compatible with the essential components of the compositionand do not otherwise unduly impair product performance, stability oraesthetics. Non-limiting examples of such counterions include halides(e.g., chloride, fluoride, bromide, iodide), sulfate and methylsulfate.

The polycation can be a synthetic polymer or naturally-occurring (i.e.,produced from organisms). In one aspect, when the polycation isnaturally-occurring, the polycation is a positively-charged proteinproduced from P. californica. FIGS. 2-6 show the protein sequences ofseveral cement proteins produced by P. californica (Zhao et al. “CementProteins of the tube building polychaete Phragmatopoma californica” J.Biol. Chem. (2005) 280: 42938-42944). Table 1 provides the amino acidmole % of each protein. Referring to FIGS. 2-5, Pc1, Pc2, and Pc4-Pc8are polycations, where the polymers are cationic at neutral pH. The typeand number of amino acids present in the protein can vary in order toachieve the desired solution properties. For example, referring to Table1, Pc1 is enriched with lysine (13.5 mole %) while Pc4 and Pc5 areenriched with histidine (12.6 and 11.3 mole %, respectively).

In the case when the polycation is a synthetic polymer, a variety ofdifferent polymers can be used; however, it is desirable that thepolymer be biocompatible and non-toxic to cells and tissue. In oneaspect, the polycation includes a polyacrylate having one or morependant amino groups. For example, the backbone can be a homopolymer orcopolymer derived from the polymerization of acrylate monomersincluding, but not limited to, acrylates, methacrylates, acrylamides,and the like. In one aspect, the backbone of the polycation ispolyacrylamide. In other aspects, the polycation is a block co-polymer,where segments or portions of the co-polymer possess cationic groupsdepending upon the selection of the monomers used to produce theco-polymer.

In one aspect, the polycation is a polyamino compound. In anotheraspect, the polyamino compound has 10 to 90 mole % tertiary aminogroups. In a further aspect, the polycation polymer has at least onefragment of the formula I

wherein R¹, R², and R³ are, independently, hydrogen or an alkyl group, Xis oxygen or NR⁵, where R⁵ is hydrogen or an alkyl group, and m is from1 to 10, or the pharmaceutically-acceptable salt thereof. In anotheraspect, R¹, R², and R³ are methyl and m is 2. Referring to formula I,the polymer backbone is composed of —CH₂—C(R¹)—C(O)X—, which is aresidue of an acrylate, methacrylate, acrylamide, or methacrylamide. Theremaining portion of formula I (CH₂)_(m)—NR²R³ is the pendant aminogroup. FIG. 3 (structures C and D) and FIG. 6 (4 and 7) show examples ofpolycations having the fragment of formula I, where the polymer backboneis composed acrylamide and methacrylate residues. In one aspect, thepolycation is the free radical polymerization product of a cationictertiary amine monomer (2-dimethylamino-ethyl methacrylate) andacrylamide, where the molecular weight is from 10 to 20 kd and possessestertiary monomer concentrations from 15 to 30 mol %. FIG. 4 (structuresE and F) and FIG. 6 (5) provide examples of polycations useful herein,where imidazole groups are directly attached to the polymer backbone(structure F) or indirectly attached to the polymer backbone via alinker (structure E via a methylene linker).

Similar to the polycation, the polyanion can be a synthetic polymer ornaturally-occurring. In one aspect, when the polyanion isnaturally-occurring, the polyanion is a negatively-charged proteinproduced from P. californica. FIGS. 2 and 7 show the sequences of twoproteins (Pc3a and Pc3b) produced by P. californica (Zhao et al. “CementProteins of the tube building polychaete Phragmatopoma californica” J.Biol. Chem. (2005) 280: 42938-42944). Referring to Table 1, Pc3a andPc3b are essentially composed of polyphosphoserine, which is anionic atneutral pH.

When the polyanion is a synthetic polymer, it is generally anybiocompatible polymer possessing anionic groups or groups that can bereadily converted to anionic groups by adjusting the pH. Examples ofgroups that can be converted to anionic groups include, but are notlimited to, carboxylate, sulfonate, phosphonate, boronate, sulfate,borate, or phosphate. Any cationic counterions can be used inassociation with the anionic polymers if the considerations discussedabove are met.

In one aspect, the polyanion is a polyphosphate. In another aspect, thepolyanion is a polyphosphate compound having from 10 to 90 mole %phosphate groups. In a further aspect, the polyanion includes apolyacrylate having one or more pendant phosphate groups. For example,the backbone can be a homopolymer or copolymer derived from thepolymerization of acrylate monomers including, but not limited to,acrylates, methacrylates, acrylamides, and the like. In one aspect, thebackbone of the polyanion is polyacrylamide. In other aspects, thepolyanion is a block co-polymer, where segments or portions of theco-polymer possess anionic groups depending upon the selection of themonomers used to produce the co-polymer. In a further aspect, thepolyanion can be heparin sulfate, hyaluronic acid, chitosan, and otherbiocompatible and biodegradable polymers typically used in the art.

In one aspect, the polyanion is a polyphosphate. In another aspect, thepolyanion is a polymer having at least one fragment having the formulaII

wherein R⁴ is hydrogen or an alkyl group, and n is from 1 to 10, or thepharmaceutically-acceptable salt thereof. In another aspect, wherein R⁴is methyl and n is 2. Similar to formula I, the polymer backbone offormula II is composed of a residue of an acrylate or methacrylate. Theremaining portion of formula II is the pendant phosphate group. FIG. 7(structure B), shows an example of a polyanion useful herein that hasthe fragment of formula II, where the polymer backbone is composedacrylamide and methacrylate residues. In one aspect, the polyanion isthe polymerization product ethylene glycol methacrylate phosphate andacrylamide, where the molecular weight is from 10,000 to 50,000,preferably 30,000, and has phosphate groups in the amount of 45 to 90mol %.

As described above, the polycation and polyanion contain crosslinkablegroups. For example, the polyanion can include one or more groups thatcan undergo oxidation, and the polycation contains on or morenucleophiles that can react with the oxidized crosslinker to produce newcovalent bonds. Polymers 3 and 7 in FIG. 6 provide examples of DOPAresidues incorporated into a polyanion and polycation, respectively. Ineach of these polymers, an acrylate containing the pendant DOPA residueis polymerized with the appropriate monomers to produce the polyanion 3and polycation 7 with pendant DOPA residues.

It is contemplated that the polycation can be a naturally occurringcompound (e.g., protein from P. californica) and the polyanion is asynthetic compound. In another aspect, the polycation can be a syntheticcompound and the polyanion is a naturally occurring compound (e.g.,protein from P. californica). In a further aspect, both the polyanionand polycation are synthetic compounds.

The adhesive complex coacervates also contain one or more multivalentcations (i.e., cations having a charge of +2 or greater). In one aspect,the multivalent cation can be a divalent cation composed of one or morealkaline earth metals. For example, the divalent cation can be a mixtureof Ca⁺² and Mg⁺². In other aspects, tranition metal ions with a chargeof +2 or greater can be used as the multivalent cation. In addition tothe pH, the concentration of the multivalent cations can determine therate and extent of coacervate formation. Not wishing to be bound bytheory, weak cohesive forces between particles in the fluid may bemediated by multivalent cations bridging excess negative surfacecharges. The amount of multivalent cation used herein can vary. In oneaspect, the amount is based upon the number of anionic groups andcationic groups present in the polyanion and polycation. In theExamples, the selection of the amount of multivalent cations withrespect to producing adhesive complex coacervates and other physicalstates is addressed.

The adhesive complex coacervate can be synthesized a number of differentways. In one aspect, the polycation, the polyanion, and at least onemultivalent cation, can be mixed with one another to produce theadhesive complex coacervate. By adding the appropriate amount ofmultivalent cation to the mixture of polyanion and polycation, theadhesive complex coacervate can be produced. In another aspect, theadhesive complex coacervate can be produced by the process comprising:

(a) preparing a polyelectrolyte complex comprising admixing at least onepolycation, at least one polyanion, and at least one multivalent cation,wherein at least one polycation or polyanion is a synthetic compound,and the polycation and/or polyanion comprises at least one group capableof crosslinking with each other; and(b) adjusting the pH of the polyelectrolyte complex, the concentrationof at least one multivalent cation, or a combination thereof to producethe adhesive complex coacervate.In this aspect, the polyelectrolyte complex is converted to the adhesivecomplex coacervate. Methods for producing the adhesive complexcoacervate in situ are described below.

The adhesive complex coacervates described herein have numerous benefitswith respect to their use as biological cements and delivery devices.For example, the coacervates have low initial viscosity, specificgravity greater than one, and being mostly water by weight, lowinterfacial tension in an aqueous environment, all of which contributeto their ability to adhere to a wet surface. An additional advantagewith respect to the bonding mechanism (i.e., crosslinking) of theadhesive complex coacervates includes low heat production duringsetting, which prevents damage to living tissue. The components can bepre-polymerized in order to avoid heat generation by in situ exothermicpolymerization. This is due for the most part by the ability of theadhesive complex coacervates to crosslink intermolecularly under verymild conditions as described above.

The adhesive complex coacervates described herein can be applied to anumber of different biological substrates. The substrate can becontacted in vitro or in vivo. The rate of crosslinking within theadhesive complex coacervate can be controlled by for example pH and thepresence of an oxidant or other agents that facilitate crosslinking. Oneapproach for applying the adhesive complex coacervate to the substratecan be found in FIG. 9. The techniques depicted in FIG. 9 are referredto herein as “spot welding,” where the adhesive complex coacervate isapplied at distinct and specific regions of the substrate. In oneaspect, the adhesive complex coacervate can be produced in situ.Referring to FIG. 9A, a pre-formed stable PEC solution 1 composed ofpolycations and polyanions at low pH (e.g., 5) is simultaneously appliedto a substrate with a curing solution 2 composed of an oxidant at ahigher pH (e.g., 10) with the use of syringes. Upon mixing, the curingsolution simultaneously produces the adhesive complex coacervate bycrosslinking the polymers on the surface of the substrate.

In another aspect, referring to FIG. 9B, a solution of polyanions 3 andpolycations 4 are applied simultaneously to the substrate. One of thesolutions has a pH higher than the other in order to produce theadhesive complex coacervate. Referring to FIG. 9B, polyanion 3 is at alower pH than the polycation solution 4; however, it is alsocontemplated that the polyanion can be in solution having a higher pHthan the polycation. The solution having the higher pH can include anoxidant in order to facilitate crosslinking.

FIG. 9C depicts another aspect of spot welding. In this aspect, thesubstrate is primed with polycation at a particular pH. Next, a solutionof the polyanion at a higher pH is applied to the polycation in order toproduce the adhesive complex coacervate in situ. It is also contemplatedthat the substrate can be primed with polyanion first followed bypolycation. An oxidant can then be applied separately on the complexcoacervate to facilitate crosslinking to produce the adhesive complexcoacervate. Alternatively, the solution applied after the substrate hasbeen primed can contain the oxidant so that the adhesive complexcoacervate is formed and subsequently crosslinked in situ.

The adhesive complex coacervates described herein can be used to repaira number of different bone fractures and breaks. The coacervates adhereto bone (and other minerals) through several mechanisms (see FIG. 1C).The surface of the bone's hydroxyapatite mineral phase (Ca₅(PO₄)₃(OH))is an array of both positive and negative charges. The negative groupspresent on the polyanion (e.g., phosphate groups) can interact directlywith the positive surface charges or it can be bridged to the negativesurface charges through the cationic groups on the polycation and/ormultivalent cations. Likewise, direct interaction of the polycation withthe negative surface charges would contribute to adhesion. Additionally,when the polycation and/or polyanion contain catechol moieties, they canfacilitate the adhesion of the coacervate to readily wet hydroxyapatite.Other adhesion mechanisms include direct bonding of unoxidizedcrosslinker (e.g., DOPA or other catechols) to hydroxyapatite.Alternatively, oxidized crosslinkers can couple to nucleophilicsidechains of bone matrix proteins.

Examples of such breaks include a complete fracture, an incompletefracture, a linear fracture, a transverse fracture, an oblique fracture,a compression fracture, a spiral fracture, a comminuted fracture, acompacted fracture, or an open fracture. In one aspect, the fracture isan intra-articular fracture or a craniofacial bone fracture. Fracturessuch as intra-articular fractures are bony injuries that extend into andfragment the cartilage surface. The adhesive complex coacervates may aidin the maintenance of the reduction of such fractures, allow lessinvasive surgery, reduce operating room time, reduce costs, and providea better outcome by reducing the risk of post-traumatic arthritis.

In other aspects, the adhesive complex coacervates described herein canbe used to join small fragments of highly comminuted fractures. In thisaspect, small pieces of fractured bone can be adhered to an existingbone. It is especially challenging to maintain reduction of the smallfragments by drilling them with mechanical fixators. The smaller andgreater number of fragments the greater the problem. In one aspect, theadhesive complex coacervate or precursor thereof may be injected insmall volumes to create spot welds as described above in order to fixthe fracture rather than filling the entire crack. The smallbiocompatible spot welds would minimize interference with healing of thesurrounding tissue and would not necessarily have to be biodegradable.In this respect it would be similar to permanently implanted hardware.

In other aspects, the adhesive complex coacervates can be used to securescaffolds to bone and other tissues such as, for example, cartilage,ligaments, tendons, soft tissues, organs, and synthetic derivatives ofthese materials. Using the complexes and spot welding techniquesdescribed herein, the development of scaffolds is contemplated. Smalladhesive tacks composed of the adhesive complex coacervates describedherein would not interfere with migration of cells or transport of smallmolecules into or out of the scaffold. In certain aspects, the scaffoldcan contain one or more drugs that facilitate growth or repair of thebone and tissue. For example, the scaffold can be coated with the drugor, in the alternative, the drug can be incorporated within the scaffoldso that the drug elutes from the scaffold over time.

The adhesive complex coacervates and methods described herein havenumerous dental applications. For example, the adhesive complexcoacervates can be used to repair breaks or cracks in teeth, forsecuring crowns, or seating implants and dentures. Using the spot weldtechniques described herein, the adhesive complex coacervate orprecursor thereof can be applied to a specific points in the mouth(e.g., jaw, sections of a tooth) followed by attaching the implant tothe substrate.

In other aspects, the adhesive complex coacervates can adhere a metalsubstrate to bone. For example, implants made from titanium oxide,stainless steel, or other metals are commonly used to repair fracturedbones. The adhesive complex coacervate or a precursor thereof can beapplied to the metal substrate, the bone, or both prior to adhering thesubstrate to the bone. In certain aspects, the crosslinking grouppresent on the polycation or polyanion can form a strong bond withtitanium oxide. For example, it has been shown that DOPA can stronglybind to wet titanium oxide surfaces (Lee et al., PNAS 103:12999 (2006)).Thus, in addition to bonding bone fragments, the adhesive complexcoacervates described herein can facilitate the bonding of metalsubstrates to bone, which can facilitate bone repair and recovery.

It is also contemplated that the adhesive complex coacervates describedherein can encapsulate one or more bioactive agents. The bioactiveagents can be any drug that will facilitate bone growth and repair whenthe complex is applied to the bone. The rate of release can becontrolled by the selection of the materials used to prepare the complexas well as the charge of the bioactive agent if the agent is a salt.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, and methods described and claimed herein aremade and evaluated, and are intended to be purely exemplary and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.) but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C. or is at ambienttemperature, and pressure is at or near atmospheric. There are numerousvariations and combinations of reaction conditions, e.g., componentconcentrations, desired solvents, solvent mixtures, temperatures,pressures and other reaction ranges and conditions that can be used tooptimize the product purity and yield obtained from the describedprocess. Only reasonable and routine experimentation will be required tooptimize such process conditions.

Mimetic Copolymer Synthesis and Characterization.

Pc3 analogs. The dopa analog monomer (dopamine methacrylamide, DMA) wasprepared by slight modification of a published procedure. (Lee B P,Huang K, Nunalee F N, Shull K R, Messersmith P B. Synthesis of3,4-dihydroxyphenylalanine (DOPA) containing monomers and theirco-polymerization with PEG-diacrylate to form hydrogels. J Biomater SciPolym Ed 2004; 15(4):449-464). Briefly, a borate-dopamine complex wasreacted at pH>9 with methacryloyl chloride. After disrupting theborate-catechol bond by acidification, the product was washed with ethylacetate, recrystallized from hexane, and verified by ¹H NMR (400 MHz,DMSO-TMS): d8.8-8.58 (2H, (OH)₂—Ar—), 7.92 (1H, —C(═O)—NH—), 6.64-6.57(2H, C₆H₂(OH)₂—), 6.42 (1H, C₆H₂H(OH)₂—), 5.61 (1H, —C(═O)—C(—CH₃)═CHH),5.30 (1H, —C(═O)—C(—CH₃)═CHH), 3.21 (2H, C₆H₃(OH)₂—CH₂—CH₂(NH)—C(═O)—),2.55 (2H, C₆H₃(OH)₂—CH₂—CH₂(NH)—C(═O)—), 1.84 (3H, —C(═O)—C(—CH₃)═CH₂).

Before polymerization monoacryloxyethyl phosphate (MAEP, Polysciences)was diluted in MeOH and extracted with hexane to remove dienes.Copolymer 1 was prepared by mixing 90 mol % MAEP, 8 mol % DMA, 2 mol %acrylamide (Aam, Polysciences), and 0.1 mol % FITC-methacrylamide inMeOH at a final monomer concentration of 5 wt %. Free radicalpolymerization was initiated with azobisisobutyronitrile (AIBN) andproceeded at 60° C. for 24 hrs in sealed ampules. A similar procedurewas used to make polymers 3-7 as shown in FIGS. 2-7. Copolymer 1 (FIG.10) was recovered by size exclusion chromatography (SEC) in MeOH on aSephadex LH-20 column (Sigma-Aldrich), concentrated by rotaryevaporation, dissolved in DI water, and freeze dried.

The MW and polydispersity index (PDI) of 1 were determined by SEC in DMFon a PLgel column (Polymer Labs) connected to a small angle lightscattering detector (Brookhaven BI-MWA) and refractive index monitor(Brookhaven BI-DNDC). The column was calibrated with polystyrenestandards. The MW of 1 was 245 kda with a PDI of 1.9. The dopaminesidechain concentration and reactivity was verified by UV/VISspectroscopy (e₂₈₀=2600 M⁻¹cm⁻¹). The phosphate sidechain concentrationwere determined by titration with 0.005 M NaOH using an automatedtitrator (Brinkmann Titrando 808). The UV/vis spectrum of 1 contained asingle absorption peak at 280 nm characteristic of the catechol form ofdopamine (FIG. 10B). Addition of a 1:1 molar ratio of NaIO₄ to 1 at pH5.0 oxidized the dopa catechol to dopaquinone with an absorption peaknear 395 nm as expected. The dopaquinone peak was stable for several hrsat pH<5.

Pc1 analogs. The lysine sidechains of Pc1 were mimicked withN-(3-aminopropyl)methacrylamide hydrochloride (APMA, Polysciences).Copolymer 2 (FIG. 10) was synthesized by dissolving 10 mol % APMA and 90mol % Aam in DI water, degassing with N₂ and initiating polymerizationwith 2 mol % ammonium persulfate (Polysciences). Polymerizationproceeded at 50° C. for 24 hrs in sealed ampules. Polymer was recoveredby dialysis against water for 3 days, and then freeze dried. The primaryamine sidechain mol % was determined by ¹H NMR (400 MHz, DMSO-TMS) fromthe ratios of d 13.45 (3H, —CH3) and d 51.04 (1H, RC(═O)CHR2). The MWand PDI of 2 were determined by SEC in PBS (20 mM PO₄, 300 mM NaCl, pH7.2) on a Superose 6 column (Pharmacia). The column was calibrated withpoly-2-hydroxypropyl methacrylate standards. The MW of 2 was 165 kd andPDI was 2.4.Coacervate formation and characterization. A 5 wt % aqueous solution of2 was added dropwise while stirring to a 5 wt % aqueous solution of 1until reaching the target amine/phosphate ratio. Total copolymerconcentration was 50 mg/ml. After mixing for 30 min the pH was adjustedwith NaOH (6M). Compositions at pH (<4) conducive to polyelectrolytecomplex (PEC) formation were diluted to 1 mg/ml in DI H₂0 and the zetapotentials and size distribution of PECs were measured on a Zeta-Sizer3000HS (Malvern Instruments). At higher pH, coacervated compositionswere centrifuged at 2500 rpm in a microfuge (Eppendorf), at 25° C. for 2min to collect the coacervate phase. The volume of both phases wasmeasured. The coacervate phases were freeze dried and weighed todetermine their mass and concentration.

The phase behavior of 1 and 2 mixed at a 1:1 molar ratio of phosphate toamine sidechains (50 mg/ml combined concentration) over the pH range3-10 is shown in FIG. 11A. The calculated net copolymer chargenormalized to the total ionizable sidechain concentration is shown inFIG. 11B. Ascorbate, a reductant, was added at a 1:5 molar ratio to dopato retard oxidation of dopa by O₂ and subsequent crosslinking atelevated pH. At low pH, the polyelectrolytes formed a stable milkysolution of colloidal polyelectrolyte complexes (PECs). The meandiameter of the PECs at pH 2.1, determined by dynamic light scattering,was 360 nm with a narrow dispersity and increased to 1080 nm at pH 4.0(FIG. 11C). The crossover of the zeta potential from positive tonegative at pH 3.6 fit well with the calculated pH dependent net chargeof the complexes (FIG. 11B). The particle size could not be measuredaccurately above pH 4 because the complexes flocculated. As the netcharge increased due to the deprotonation of the phosphate sidechains,the copolymers condensed into a dense second phase. At pH 5.1 theseparated phase had the character of a loose low density precipitate. AtpH 7.2 and 8.3 the dense phase had the character of a cohesive liquidcomplex coacervate (FIG. 12). The copolymers were concentrated aboutthree-fold to 148 and 153 mg/ml, respectively, in the coacervatedphases. At pH 9.5 the polyelectrolyte mixture formed a dense non-liquidionic gel. At pH 10 the copolymers went into solution and spontaneouslycrosslinked through the dopaquinone and amine sidechains into a clearhydrogel.

Extraction of divalent cations with the chelator EDTA resulted in a 50%decrease in compressive strength of P. californica tubes, a ten-folddecrease in adhesiveness, and collapse of the glues porous structure.The effect of divalent cations on the phase behavior of the mimeticpolyelectrolytes was investigated by mixing 1 and 2 at amine tophosphate sidechain ratios ranging from 1:1 to 0:1 with divalent cationto phosphate sidechain ratios ranging from 0:1 to 1:1 to create acoacervate phase diagram (FIG. 13). The pH was fixed at 8.2, the pH ofseawater, and divalent cations were added as a 4:1 mixture of Mg²⁺ andCa²⁺, the approximate Mg²⁺/Ca²⁺ ratio in the natural glue determined byelemental analysis. The highest mass of coacervate (dark gray squares)occurred in mixtures with higher amine to phosphate sidechain ratios andlower divalent cation to phosphate sidechain ratios. Mixtures with lowerpolyamine ratios were clear (clear squares) even at higher divalentcation/phosphate sidechain ratios. At higher amine/phosphate anddivalent cation/phosphate ratios the solutions were turbid (light graysquares) with slight precipitates but much less turbid than solutionscontaining PECs (medium gray squares).

Mechanical bond testing. Bone test specimens, ˜1 cm³, were cut with aband saw from bovine femur cortical bone, obtained from a local grocerystore, sanded with 320 grit sandpaper, and stored at −20° C. NaIO₄ at a1:2 molar ratio to dopa sidechains was evenly applied to one face eachof two wet bone specimens. Forty ml, a volume sufficient to completelyfill the space between 1 cm² bone interfaces, of the test coacervatesolution was applied with a pipette, the bone specimens were pressedtogether squeezing out a small excess of adhesive, clamped, andimmediately wrapped in PBS (20 mM PO₄, 150 mM NaCl, pH 7.4) soakedgauze. The applied coacervate contained ascorbate at a 1:5 molar ratioto dopa to prevent premature crosslinking. The bonded specimens wereincubated at 37° C. for at least 24 hr in a sealed container containingsoaked sponges to maintain 100% humidity. Reference specimens werebonded with 40 ml Loctite 401 superglue in exactly the same manner. Acommercial non-medical grade cyanoacrylate was used because there are nohard tissue medical adhesives available for comparison. Mechanical testswere performed on a custom built material testing system using a 1 kgload cell. The instrument was controlled and data acquired using LabView(National Instruments). One bone of a bonded pair was clamped laterally1 mm from the bond interface. The second bone was pressed with across-head speed of 0.02 mm/s against a dull blade positioned 1 mmlateral to the bond interface. Bond strength tests were performed atroom temperature immediately after unwrapping the wet specimens toprevent drying. After testing, the bonds were examined for failure mode.The bonded area was measured by tracing an outline of the bone contactsurface on paper, cutting out the trace, and determining its area fromthe weight of the paper cut-out. At least 6 specimens were tested foreach condition.

The shear modulus and strength at failure were measured with bovinecortical bone specimens bonded while wet with the three coacervatingcompositions marked with an asterisk in FIG. 13. The coacervate densityin the three compositions increased with increasing divalent cationratios (to 120, 125, and 130 mg/ml, respectively). Both the modulus andbond strength of the fully hydrated specimens increased with increasingdivalent cation concentration, reaching 37% of the strength of wet bonesbonded with a commercial cyanoacrylate adhesive (FIG. 14A). Thecyanoacrylate adhesive was used as a reference point because there areno bone adhesives in clinical use for comparison. The strength of themimetic adhesive is also about {grave over (1)}{grave over (/)}{graveover (3)} the strength of natural P. californica glue estimated to be350 kPa and mussel byssal glue estimated to range from 320 to 750 kPadependent on the season. In almost all cases the bonds failed cohesivelyleaving adhesive on both bone interfaces, which suggested thecompositions formed strong interfacial bonds with hydroxyapatite. Thebonds were dimensionally stable, neither shrinking nor swellingappreciably after complete submersion in PBS pH 7.2 for several months(FIG. 14B). Dimensional stability during cure and long term exposure towater is an important requirement for a useful bone adhesive.

Dopamine-mediated copolymer crosslinking. Addition of NaIO₄ to solutionsof 3 at a 1:1 molar ratio immediately and quantitatively oxidized DOPA(280 nm) to dopaquinone (392 nm). Within a few minutes the quinine peakdecayed into broad general absorption as the reactive quinones formedcovalent diDOPA crosslinks (FIG. 15, top left). Crosslinking between thequinones and primary amines (FIG. 15, bottom left) led to a broadergeneral absorption than diDOPA crosslinking Dopamine oxidation andcrosslinking chemistry therefore behaved as expected in the dopaminecopolymers. The dopamine copolymers rapidly formed hydrogels as a resultof oxidative crosslinking (FIGS. 15, A&C). Oxidized phosphodopamine 3did not gel by itself (FIG. 15B) but when mixed with 4 it gelled rapidly(FIG. 15D). Intermolecular diDOPA crosslinking between PO₄ copolymerswas inhibited but not intermolecular DOPA-amine crosslinking Thisprovides a crosslinking control mechanism that may be useful forformulating and delivering a synthetic adhesive.pH triggered DOPA-mediated crosslinking. To explore, the pH dependenceand kinetics of DOPA oxidation, crosslinking of the dopamine copolymerswere evaluated by UV-Vis spectroscopy. Results with p(EGMP[92]-DMA[8])(3) are shown in FIG. 16. UV-vis spectra were acquired at increasingtime after addition of a stoichiometric amount of NaIO₄. At pH 5.0(top), dopaquinone absorbance (398 nm) was maximal in ˜15 min andremained stable for several hrs (inset). At pH 6.0, absorbance at 398 nmpeaked in <1 min and evolved into broad absorbance with peaks at 310 and525 nm. The broad absorbance is not due to dopaquinone crosslinkingsince gels do not form (FIG. 16). For comparison, 6 was oxidized at lowpH crosslinked but at a significantly slower rate (not shown).

The results show that the dopaquinone is stable at low pH and diDOPAcrosslinking was inhibited at higher pH in the phosphodopaminecopolymers. In the presence of the polyamine, the covalent crosslinkingwas channeled toward intermolecular amine-DOPA bonds. This is animportant observation because it lays out a path to controlled deliveryand setting of the synthetic adhesive.

In vitro cytotoxicity. Solutions of 3 and 4, 40 wt % each, were mixed atlow pH to form a polyelectrolyte complex. The solution was partiallyoxidized with NaIO₄ and basified with NaOH just before application tosterile glass coverslips. The adhesive-treated coverslips were placed inthe bottom of culture plate wells and human foreskin fibroblasts, humantracheal fibroblasts, and rat primary astrocytes in serum containingmedia were added to separate wells at 30K cells/well (FIG. 17). After 24hr, the cells were fixed with 4% para-formaldehdye, then immunostainedfor the intermediate filament protein, vimentin, to visualize cellmorphology (green, A-B), pericellular fibronectin to assess ECMsecretion (red, B), glial fibrillary protein to visual primary astrocytemorphology (green, C), and DAPI to visualize nuclei (blue, C). Thegranular globs of adhesive auto-fluoresced orangish-red (A-C).

In the representative images (FIG. 17), all cell types had morphologiesindistinguishable from cells growing on glass without adhesive. Thecells had normal motility and in several cases extended processes thatdirectly contacted the adhesive. No toxicity was apparent.

Rat calvarial defect model. Production of the fragmented defect andrepair with an adhesive complex coacervate is shown in FIGS. 18A-F. MaleSprague Dawley rats (256-290 g) (Harlan) were anesthetized with amixture of ketamine (65 mg/kg), xylazine (7.5 mg/kg), and acepromazine(0.5 mg/kg). At full depth of anesthesia, the eyes were covered withophthalmic ointment, the head shaved, and the scalp disinfected withisopropanol and butadiene. With the prepped rats in a stereotacticframe, a compressed air-driven drill operating at ˜5000 RPM was loweredusing a stereotactic fine toothed manipulator. Sterile saline or PBS wascontinuously applied at the craniotomy site while the custom madetrephine tool was lowered 600 microns (previously determined as theskull thickness of rats the age of which were used in the experiment).The result is a round, accurate hole through the skull with littleobservable effect on the underlying dura or vasculature (FIG. 18A-B).The bone plug was recovered with fine curved forceps and broken intofragments using a hemostat and fine rongeur (FIG. 18B). The bonefragments were returned to the defect (FIG. 18C) and 5 μl of testadhesive (3 and 4 mixed immediately prior to the application of thefracture) was applied with a micropipettor (FIG. 18D). The low viscosityadhesive solution (pre-formed PECS mixed with curing solution justbefore delivery) readily and cleanly wicked into the fractures. Within 5min the fragments were sufficiently fixed that they could be tappedsharply with the forceps without displacement. The adhesive continued toturn dark reddish brown as it cured (FIG. 18E-F).

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the compounds, compositions and methods described herein.

Various modifications and variations can be made to the compounds,compositions and methods described herein. Other aspects of thecompounds, compositions and methods described herein will be apparentfrom consideration of the specification and practice of the compounds,compositions and methods disclosed herein. It is intended that thespecification and examples be considered as exemplary.

1. An adhesive complex coacervate comprising at least one polycation, atleast one polyanion, and at least one multivalent cation, wherein atleast one polycation or polyanion is a synthetic compound, and thepolycation and/or polyanion are crosslinked with one another uponcrosslinking.
 2. The coacervate of claim 1, wherein the polycationcomprises a polyamino compound.
 3. The coacervate of claim 1, whereinthe polycation comprises a polyacrylate comprising one or more pendantamino groups.
 4. The coacervate of claim 1, wherein the polycationcomprises a polyacrylate comprising one or more pendant imidazolegroups.
 5. The coacervate of claim 1, wherein the polycation comprises apolymer comprising at least one fragment comprising the formula I

wherein R¹, R², and R³ are, independently, hydrogen or an alkyl group, Xis oxygen or NR⁵, where R⁵ is hydrogen or an alkyl group, and m is from1 to 10, or the pharmaceutically-acceptable salt thereof.
 6. Thecoacervate of claim 5, wherein R¹, R², and R³ are methyl, X is NH, and mis
 2. 7. The coacervate of claim 1, wherein the polycation comprises apositively-charged protein produced from P. californica.
 8. Thecoacervate of claim 2, wherein the polyamino compound comprises from 10to 90 mole % primary, secondary, or tertiary amino groups.
 9. Thecoacervate of claim 1, wherein the polyanion comprises one or moresulfate, sulfonate, carboxylate, borate, boronate, phosphonate, orphosphate groups.
 10. The coacervate of claim 1, wherein the polyanioncomprises a polyphosphate compound.
 11. The coacervate of claim 1,wherein the polyanion comprises a polyacrylate comprising one or morependant phosphate groups.
 12. The coacervate of claim 1, wherein thepolyanion comprises a polymer comprising at least one fragmentcomprising the formula II

wherein R⁴ is hydrogen or an alkyl group, and n is from 1 to 10, or thepharmaceutically-acceptable salt thereof.
 13. The coacervate of claim12, wherein R⁴ is methyl and n is
 2. 14. The coacervate of claim 11,wherein the polyphosphate compound comprises from 10 to 90 mole %phosphate groups.
 15. The coacervate of claim 1, wherein the polyanioncomprises a polyphosphoserine.
 16. The coacervate of claim 1, whereinthe polyanion comprises a polyphosphoserine produced from the P.californica gene.
 17. The coacervate of claim 1, wherein the multivalentcation comprises one or more transition metal ions or rare earth metals.18. The coacervate of claim 1, wherein the multivalent cation comprisesone or more divalent cations.
 19. The coacervate of claim 1, wherein themultivalent cation comprises Ca⁺² and Mg⁺².
 20. The coacervate of claim1, wherein the composition is biocompatible and biodegradable.
 21. Thecoacervate of claim 1, wherein the composition further comprises one ormore bioactive agents encapsulated in the complex.
 22. The coacervate ofclaim 1, wherein the groups capable of crosslinking with one another arethe same or different.
 23. The coacervate of claim 1, wherein thecrosslinking group on the polycation comprises a nucleophilic group andthe crosslinking group on the polyanion comprises an electrophilicgroup.
 24. The coacervate of claim 1, wherein the crosslinking group onthe polycation and polyanion comprises a hydroxyl aromatic compoundcapable of undergoing oxidation.
 25. The coacervate of claim 1, whereinthe crosslinking group on the polyanion comprises a DOPA residue or acatechol residue and the polycation comprises a nucleophilic groupcapable of reacting with the crosslinking group to form a covalent bond.26. An adhesive complex coacervate produced by the process comprising(a) preparing a polyelectrolyte complex comprising admixing at least onepolycation, at least one polyanion, and at least one multivalent cation,wherein at least one polycation or polyanion is a synthetic compound,and the polycation and/or polyanion comprises at least one group capableof crosslinking with each other; and (b) adjusting the pH of thepolyelectrolyte complex, the concentration of the at least one divalentcation, or a combination thereof to produce the adhesive complexcoacervate.
 27. The coacervate of claim 26, wherein the coacervate isproduced in situ.
 28. The coacervate of claim 26, wherein step (a) isperformed at a pH less than 4, and step (b) comprises raising the pH.29. The coacervate of claim 26, wherein step (b) comprises raising thepH of the polyelectrolyte complex to a pH of greater than or equal to7.0.
 30. The coacervate of claim 26, wherein after step (b) furthercomprises contacting the complex coacervate with an oxidant in order tofacilitate the crosslinking between the polycation and polyanion. 31.The coacervate of claim 30, wherein the oxidant comprises O₂, NaIO₄, aperoxide, or a transition metal oxidant.
 32. A polyelectrolyte complexcomprising at least one polycation, at least one polyanion, and at leastone multivalent cation, wherein at least one polycation or polyanion isa synthetic compound, and the polycation and/or polyanion comprises atleast one group capable of crosslinking with each other.
 33. A complexcoacervate comprising at least one polycation, at least one polyanion,and at least one multivalent cation, wherein at least one polycation orpolyanion is a synthetic compound, and the polycation and/or polyanioncomprises at least one group capable of crosslinking with each other.34. A method for applying an adhesive complex coacervate to a substrate,the method comprising applying simultaneously to the substrate (1) thepolyelectrolyte complex of claim 26 having a first pH and (2) a solutioncomprising an oxidant having a second pH, wherein the second pH isgreater than the first pH.
 35. A method for applying an adhesive complexcoacervate to a substrate, the method comprising applying simultaneouslyto the substrate (1) a first solution comprising a polycation having afirst pH and (2) a second solution comprising a polyanion having asecond pH, wherein (i) when the first pH is less than 7, the second pHis greater than 7; (ii) when the first pH is greater than 7, the secondpH is less than 7; (iii) wherein at least one polycation or polyanion isa synthetic compound; (iv) the polyanion comprises at least one groupcapable of crosslinking with the polycation; (v) the first solutionand/or the second solution comprises at least one multivalent cation;and (vi) and the first solution and/or second solution comprises anoxidant, wherein upon mixing of the first solution and second solutionthe polyanion and polycation crosslink with one another to produce theadhesive complex coacervate.
 36. A method for applying an adhesivecomplex coacervate to a substrate, the method comprising (1) applying tothe substrate a first solution comprising a polycation followed by (2)applying a second solution comprising a polyanion and an oxidant,wherein (i) at least one polycation or polyanion is a syntheticcompound; (ii) the polyanion comprises at least one group capable ofcrosslinking with the polycation; (iii) the first solution and/or thesecond solution comprises at least one multivalent cation; and the pH ofthe second solution is greater than the pH of the first solution,wherein the polyanion and polycation crosslink with one another toproduce the adhesive complex coacervate.
 37. A method for applying anadhesive to a substrate, the method comprising (1) applying to thesubstrate a first solution comprising a polyanion followed by (2)applying a second solution comprising a polycation and an oxidant,wherein (i) at least one polycation or polyanion is a syntheticcompound; (ii) the polyanion comprises at least one group capable ofcrosslinking with the polycation; (iii) the first solution and/or thesecond solution comprises at least one multivalent cation; and the pH ofthe second solution is greater than the pH of the first solution,wherein the polyanion and polycation crosslink with one another toproduce the adhesive complex coacervate.
 38. The method of claim 34,wherein the substrate comprises bone or an implantable device.
 39. Amethod for repairing a bone fracture in a subject, comprising contactingthe fractured bone with the adhesive complex coacervate of claim
 1. 40.The method of claim 39, wherein the method is in vitro.
 41. The methodof claim 39, wherein the method is in vivo.
 42. The method of claim 39,wherein the fracture comprises complete fracture, an incompletefracture, a linear fracture, a transverse fracture, an oblique fracture,a compression fracture, a spiral fracture, a comminuted fracture, acompacted fracture, or an open fracture.
 43. The method of claim 39,wherein the fracture comprises an intra-articular fracture.
 44. Themethod of claim 39, wherein the fracture comprises a craniofacial bonefracture.
 45. The method of claim 39, wherein the method comprisesadhering a fractured piece of bone to an existing bone.
 46. The methodof claim 39, wherein the composition sets within 60 seconds.
 47. Amethod for adhering a metal substrate to a bone of a subject comprisingcontacting the bone with the composition of claim 1 and applying themetal substrate to the coated bone.
 48. A method for adhering abone-tissue scaffold to a bone of a subject comprising contacting thebone and tissue with the composition of claim 1 and applying thebone-tissue scaffold to the bone and tissue.
 49. The method of claim 48,wherein the tissue comprises cartilage, a ligament, a tendon, a softtissue, an organ, or synthetic derivative thereof.
 50. The method ofclaim 48, wherein the scaffold comprises one or more drugs thatfacilitate growth or repair of the bone and tissue.
 51. A method forrepairing a crack in a tooth, comprising applying the composition ofclaim 1 to the crack.
 52. A method for securing a dental implant,comprising applying the composition of claim 1 to an oral substrate andattaching the dental implant to the substrate.
 53. The method of claim52, wherein the dental implant comprises a crown or denture.
 54. Amethod for delivering one or more bioactive agents comprisingadministering the coacervate of claim 1 to a subject.
 55. A polymercomprising a (1) a polymeric backbone, (2) one or more phosphate groupslinked to the backbone, and (3) one or more hydroxyl aromatic groupslinked to the backbone capable of undergoing oxidation in the presenceof an oxidant.
 56. The polymer of claim 55, wherein the polymericbackbone is derived from the polymerization of an acrylate.